Mikko Kuokkanen

Independent Introduction of Two Lactase-Persistence Alleles into Human Populations Reflects Different History of Adaptation to Milk Culture

The T(-13910) variant located in the enhancer element of the lactase (LCT) gene correlates perfectly with lactase persistence (LP) in Eurasian populations whereas the variant is almost nonexistent among Sub-Saharan African populations, showing high prevalence of LP. Here, we report identification of two new mutations among Saudis, also known for the high prevalence of LP. We confirmed the absence of the European T(-13910) and established two new mutations found as a compound allele: T/G(-13915) within the -13910 enhancer region and a synonymous SNP in the exon 17 of the MCM6 gene T/C(-3712), -3712 bp from the LCT gene. The compound allele is driven to a high prevalence among Middle East population(s). Our functional analyses in vitro showed that both SNPs of the compound allele, located 10 kb apart, are required for the enhancer effect, most probably mediated through the binding of the hepatic nuclear factor 1 alpha (HNF1 alpha). High selection coefficient (s) approximately 0.04 for LP phenotype was found for both T(-13910) and the compound allele. The European T(-13910) and the earlier identified East African G(-13907) LP allele share the same ancestral background and most likely the same history, probably related to the same cattle domestication event. In contrast, the compound Arab allele shows a different, highly divergent ancestral haplotype, suggesting that these two major global LP alleles have arisen independently, the latter perhaps in response to camel milk consumption. These results support the convergent evolution of the LP in diverse populations, most probably reflecting different histories of adaptation to milk culture.

Identification of IL6R and chromosome 11q13.5 as risk loci for asthma

BACKGROUND We aimed to identify novel genetic variants affecting asthma risk, since these might provide novel insights into molecular mechanisms underlying the disease. METHODS We did a genome-wide association study (GWAS) in 2669 physician-diagnosed asthmatics and 4528 controls from Australia. Seven loci were prioritised for replication after combining our results with those from the GABRIEL consortium (n=26,475), and these were tested in an additional 25,358 independent samples from four in-silico cohorts. Quantitative multi-marker scores of genetic load were constructed on the basis of results from the GABRIEL study and tested for association with asthma in our Australian GWAS dataset. FINDINGS Two loci were confirmed to associate with asthma risk in the replication cohorts and reached genome-wide significance in the combined analysis of all available studies (n=57,800): rs4129267 (OR 1·09, combined p=2·4×10(-8)) in the interleukin-6 receptor (IL6R) gene and rs7130588 (OR 1·09, p=1·8×10(-8)) on chromosome 11q13.5 near the leucine-rich repeat containing 32 gene (LRRC32, also known as GARP). The 11q13.5 locus was significantly associated with atopic status among asthmatics (OR 1·33, p=7×10(-4)), suggesting that it is a risk factor for allergic but not non-allergic asthma. Multi-marker association results are consistent with a highly polygenic contribution to asthma risk, including loci with weak effects that might be shared with other immune-related diseases, such as NDFIP1, HLA-B, LPP, and BACH2. INTERPRETATION The IL6R association further supports the hypothesis that cytokine signalling dysregulation affects asthma risk, and raises the possibility that an IL6R antagonist (tocilizumab) may be effective to treat the disease, perhaps in a genotype-dependent manner. Results for the 11q13.5 locus suggest that it directly increases the risk of allergic sensitisation which, in turn, increases the risk of subsequent development of asthma. Larger or more functionally focused studies are needed to characterise the many loci with modest effects that remain to be identified for asthma. FUNDING National Health and Medical Research Council of Australia. A full list of funding sources is provided in the webappendix.

Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia

Background and aims: The mechanism of the developmental downregulation of the lactase-phlorizin hydrolase (LPH) gene underlying adult-type hypolactasia is unknown. We have determined the functional significance of the recently identified two single nucleotide polymorphisms (SNPs), C/T−13910 and G/A−22018, associated with adult-type hypolactasia by studying LPH mRNA levels in intestinal biopsy samples with different genotypes. Methods: Intestinal biopsy samples were taken from 52 patients with abdominal complaints. Hypolactasia was diagnosed by determining lactase and sucrase activities and calculating their ratio (L/S ratio). The functional effect of the C/T−13910 and G/A−22018 genotype on expression of LPH mRNA was demonstrated in patients heterozygous for the C/T−13910 and G/A−22018 polymorphism and an informative expressed SNP located in the coding region of the LPH mRNA. Reverse transcription-polymerase chain reaction followed by solid phase minisequencing was used for accessing the relative expression levels of the LPH alleles using informative SNPs located in exons 1, 2, 6, 10, 13, or 17 as markers. Results: Statistically significant differences between the three different genotypes CC−13910 GG−22018, CT−13910 GA−22018, and TT−13910 AA-22018 and their respective L/S ratios were observed. Relative quantitation of the expressed LPH alleles showed that the persistent allele represented 92 (6)% (mean (SEM), range 78–99%; n=14) of the expressed LPH mRNA. The patient with the homozygous persistent TT−13910 AA−22018, as well as hypolactasic patients with CC−13910 GG−22018, showed equal expression of both alleles (47 (1)%; n=7). Conclusions: Expression of LPH mRNA in the intestinal mucosa in individuals with T−13910 A−22018 alleles is several times higher than that found in individuals with C−13910, G−22018 alleles. These findings suggest that the two SNPs, C/T−13910 and G/A−22018, associated with adult-type hypolactasia, are associated with the transcriptional regulation of the LPH gene. The presence of the T−13910 A−22018 allele also shows significant elevation of the L/S ratio.

Genome-Wide Association Studies of Asthma in Population-Based Cohorts Confirm Known and Suggested Loci and Identify an Additional Association near HLA

Rationale Asthma has substantial morbidity and mortality and a strong genetic component, but identification of genetic risk factors is limited by availability of suitable studies. Objectives To test if population-based cohorts with self-reported physician-diagnosed asthma and genome-wide association (GWA) data could be used to validate known associations with asthma and identify novel associations. Methods The APCAT (Analysis in Population-based Cohorts of Asthma Traits) consortium consists of 1,716 individuals with asthma and 16,888 healthy controls from six European-descent population-based cohorts. We examined associations in APCAT of thirteen variants previously reported as genome-wide significant (P<5x10−8) and three variants reported as suggestive (P<5×10−7). We also searched for novel associations in APCAT (Stage 1) and followed-up the most promising variants in 4,035 asthmatics and 11,251 healthy controls (Stage 2). Finally, we conducted the first genome-wide screen for interactions with smoking or hay fever. Main Results We observed association in the same direction for all thirteen previously reported variants and nominally replicated ten of them. One variant that was previously suggestive, rs11071559 in RORA, now reaches genome-wide significance when combined with our data (P = 2.4×10−9). We also identified two genome-wide significant associations: rs13408661 near IL1RL1/IL18R1 (P Stage1+Stage2 = 1.1x10−9), which is correlated with a variant recently shown to be associated with asthma (rs3771180), and rs9268516 in the HLA region (P Stage1+Stage2 = 1.1x10−8), which appears to be independent of previously reported associations in this locus. Finally, we found no strong evidence for gene-environment interactions with smoking or hay fever status. Conclusions Population-based cohorts with simple asthma phenotypes represent a valuable and largely untapped resource for genetic studies of asthma.

A genetic test which can be used to diagnose adult-type hypolactasia in children

Background/Aims: Adult-type hypolactasia (primary lactose malabsorption) affects most of world’s human population and limits the use of fresh milk due to lactose intolerance. The diagnosis of adult-type hypolactasia has been difficult to establish because of unsatisfactory diagnostic methods. C/T-13910 single nucleotide polymorphism residing 13910 base pairs from the 5′ end of the lactase gene has been shown to be associated with lactase persistence. The aim of the study was to assess the applicability of the C/T-13910 variant as a diagnostic test for adult-type hypolactasia during childhood. Methods: Intestinal biopsies were obtained from 329 children and adolescents of African, Finnish, and other White origins aged 0.1–20 years undergoing upper gastrointestinal endoscopy because of abdominal complaints. The biopsies were assayed for lactase, sucrase, and maltase activity and genotyped for the C/T-13910 variant using polymerase chain reaction minisequencing. Results: The frequency of the C/C-13910 genotype defining lactase non-persistence was well in agreement in this study with published figures for the prevalences of adult-type hypolactasia in Africans and Whites. The C/C-13910 genotype was associated with very low lactase activity (<10 U/g protein) in the majority of children tested at 8 years of age and in every child older than 12 years of age giving a specificity of 100% and sensitivity of 93% for the genetic test. The decline of lactase activity was somewhat earlier in African compared with Finnish children with C/C-13910 genotype (p<0.03). Conclusions: Genetic test of C/T-13910 polymorphism can be used as a first stage screening test for adult-type hypolactasia.

Genetic Variants of TSLP and Asthma in an Admixed Urban Population

Background Thymic stromal lymphopoietin (TSLP), an IL7-like cytokine produced by bronchial epithelial cells is upregulated in asthma and induces dendritic cell maturation supporting a Th2 response. Environmental pollutants, including tobacco smoke and diesel exhaust particles upregulate TSLP suggesting that TSLP may be an interface between environmental pollution and immune responses in asthma. Since asthma is prevalent in urban communities, variants in the TSLP gene may be important in asthma susceptibility in these populations. Objectives To determine whether genetic variants in TSLP are associated with asthma in an urban admixed population. Methodology and Main Results Ten tag-SNPs in the TSLP gene were analyzed for association with asthma using 387 clinically diagnosed asthmatic cases and 212 healthy controls from an urban admixed population. One SNP (rs1898671) showed nominally significant association with asthma (odds ratio (OR) = 1.50; 95% confidence interval (95% CI): 1.09–2.05, p = 0.01) after adjusting for age, BMI, income, education and population stratification. Association results were consistent using two different approaches to adjust for population stratification. When stratified by smoking status, the same SNP showed a significantly increased risk associated with asthma in ex-smokers (OR = 2.00, 95% CI: 1.04–3.83, p = 0.04) but not significant in never-smokers (OR = 1.34; 95% CI: 0.93–1.94, p = 0.11). Haplotype-specific score test indicated that an elevated risk for asthma was associated with a specific haplotype of TSLP involving SNP rs1898671 (OR = 1.58, 95% CI: 1.10–2.27, p = 0.01). Association of this SNP with asthma was confirmed in an independent large population-based cohort consortium study (OR = 1.15, 95% CI: 1.07–1.23, p = 0.0003) and the results stratified by smoking status were also validated (ex-smokers: OR = 1.21, 95% CI: 1.08–1.34, p = 0.003; never-smokers: OR = 1.06, 95% CI: 0.94–1.17, p = 0.33). Conclusions Genetic variants in TSLP may contribute to asthma susceptibility in admixed urban populations with a gene and environment interaction.

Mitochondrial aspartate/glutamate carrier SLC25A12 gene is associated with autism.

Two single nucleotide polymorphisms (SNP) within Mitochondrial Aspartate/Glutamate Carrier SLC25A12 gene have recently shown to be strongly associated with autism. Here, we attempted to replicate this finding in two separate Finnish samples with autism spectrum disorders. Family‐based association analysis of two SNPs, rs2056202 and rs2292813, previously shown to be associated with autism was performed in two samples with different phenotypic characteristics. The samples included 97 families with strictly defined autism and 29 extended families with Asperger syndrome (AS). We detected association at rs2292813 (FBAT, P=0.0018) in the Finnish autism sample. In, addition other family‐based analysis methods supported this finding. By contrast, analysis of the AS sample yielded no evidence for association. This study shows further support that genetic variants within SLC25A12 gene contribute to the etiology of autism.

Transcriptional downregulation of the lactase (LCT) gene during childhood

Adult-type hypolactasia, characterised by bloating, gas formation, and diarrhoea after ingestion of lactose containing food, affects half of the world’s population.1 The molecular background of lactase non-persistence/persistence trait has been shown to associate with a single nucleotide polymorphism (SNP) C/T−13910 residing 13910 base pairs upstream from the 5′ end of the lactase (LCT) gene in an intron of the minichromosome maintenance 6 (MCM6) gene.2–4 We have demonstrated a trimodal distribution of lactase activity in the intestinal mucosa in adults, with low lactase activity (4–9 U/g protein) in those with the C/C−13910 genotype.3 The C−13910 and T−13910 allele show differential regulation of lactase promoter activity and binding capacity for the nuclear proteins in electromobility shift assay.5,6 Our recent analysis in a paediatric population demonstrated that the main time period for lactase downregulation in Finns and in Somalians is from five to 10 years of age.4 To further assess the role …

Lactase persistence and ovarian carcinoma risk in Finland, Poland and Sweden.

Ovarian carcinoma is the fourth most common cause of cancer death in women. The cause and pathogenesis of this disease has remained obscure. Galactose, the hydrolyzing product of the milk sugar lactose, has been hypothesized to be toxic to ovarian epithelial cells and consumption of dairy products and lactase persistence has been suggested to be a risk factor for ovarian carcinoma. In adults, downregulation of lactase depends on a variant C/T−13910 at the 5′ end of the lactase gene. To explore whether lactase persistence is related to the risk of ovarian carcinoma we determined the C/T−13910 genotype in a cohort of 782 women with ovarian carcinoma. The C/T−13910 genotype was defined by solid phase minisequencing from 327 Finnish, 303 Polish, 152 Swedish patients and 938 Finnish, 296 Polish and 97 Swedish healthy individuals served as controls. Lactase persistence did not associate significantly with increased risk for ovarian carcinoma in the Finnish (odds ratio [OR] = 0.77, 95% confidence interval [CI] = 0.57–1.05, p = 0.097), in the Polish (OR = 0.95, 95% CI = 0.68–1.33, p = 0.75), or in the Swedish populations (OR = 1.63, 95% CI = 0.65–4.08, p = 0.29). Our results do not support the hypothesis that lactase persistence increases the ovarian carcinoma risk. On the contrary, lactase persistence may decrease the ovarian carcinoma risk at least in the Finnish population.

Functional significance of single nucleotide polymorphisms in the lactase gene in diverse US patients and evidence for a novel lactase persistence allele at -13909 in those of European ancestry.

Objectives: Recent data from mainly homogeneous European and African populations implicate a 140-bp region 5′ to the transcriptional start site of LCT (the lactase gene) as a regulatory site for lactase persistence and nonpersistence. Because there are no studies of US nonhomogeneous populations, we performed genotype/phenotype analysis of the −13910 and −22018 LCT single nucleotide polymorphisms (SNPs) in New England children, mostly of European ancestry. Methods: Duodenal biopsies were processed for disaccharidase activities, RNA quantification by reverse transcription polymerase chain reaction (RT-PCR), allelic expression ratios by PCR, and genotyping and SNP analysis. Results were compared with clinical information. Results: Lactase activity and mRNA levels, and sucrase-to-lactase ratios of enzyme activity and mRNA, showed robust correlations with genotype. None of the other LCT SNPs showed as strong a correlation with enzyme or mRNA levels as did −13910. Data were consistent, with the −13910 being the causal sequence variant instead of −22018. Four individuals heterozygous for −13910T/C had allelic expression patterns similar to individuals with −13910C/C genotypes; of these, 2 showed equal LCT expression from the 2 alleles and a novel variant (−13909C>A) associated with lactase persistence. Conclusions: The identification of −13910C/C genotype is likely to predict lactase nonpersistence, consistent with prior published studies. A −13910T/T genotype will frequently, but not perfectly, predict lactase persistence in this mixed European-ancestry population; a −13910T/C genotype will not predict the phenotype. A long, rare haplotype in 2 individuals with −13910T/C genotype but equal allele-specific expression contains a novel lactase persistence allele present at −13909.